Method for detecting staphylococci

ABSTRACT

The present invention exploits the herein first reported, empirical observation that even though staphylococci produce the enzyme, beta-glucosidase, this genus of bacteria is not able to produce a metabolite that will enzymatically react with 5-bromo-4-chloro-3-indolyl-beta-D-glucopyranoside, a substrate commonly used to detect the presence of beta-glucosidase produced by other bacteria. In view of this unexpected observation, one embodiment of the present invention includes a selective medium containing inhibitors to enhance staphylococci growth as well as a first glucopyranoside substrate, such as 5-bromo-4-chloro-3-indolyl-beta-D-glucopyranoside, and a second phosphatase substrate, Such as 6-chloro-3-indolylphosphate or 5-bromo-6-chloro-3-indolylphosphate. In a particularly preferred embodiment of this invention, a thin film culture plate device, such as a PETRIFILM culture plate device, is prepared using a dry culture medium containing selected inhibitors, a first glucopyranoside substrate and a second phosphate substrate. When the thin film culture plate device is inoculated with a sample and then incubated for a sufficient period of time, staphylococci in the sample will produce colonies on the thin film device having a red color and other bacteria in the sample will produce colonies on the device having a blue color.

This application is a continuation of U.S. application Ser. No.08/189,182 filed Jan. 31, 1994, now U. S. Pat. No. 5,443,963.

The present invention relates to a method of detecting and/orenumerating bacteria in a sample and, more particularly, relates to amethod of identifying and/or enumerating staphylococci in a mixedbacterial population using a novel indicator system.

BACKGROUND

A variety of methods and processes are currently available to determine,identify and enumerate bacteria in different types of samples. Forexample, methodologies are available to identify and enumerate coliformbacteria, coliforms, in samples of water, food or dairy products inorder to assess the quality or potential contamination levels of thosesamples.

One approach to distinguish E. coli, a specific type of coliformbacteria commonly classified as a Gram-negative rod bacteria, from amixed population of coliforms is reported in U.S. Pat. No. 5,210,022. Inthat patent, two substrates which form contrasting insolubleprecipitates in the presence of two specific bacterial enzymes,beta-galactosidase and beta-glucuronidase, are incorporated in a testmedium. The use of these two substrates allows coliforms to bedifferentiated from E. coli because all coliform bacteria producebeta-galactosidase while only E. coli produce beta-glucuronidase. As thedifferent colonies of bacteria grow when incubated in thesubstrate-containing medium, contrasting colored precipitates formaround either growing E. coli or coliform colonies. Identification of E.coli colonies from the other coliform colonies in the mixed populationis readily made by the contrasting colors of the precipitatedsubstrates.

A need for such selective differentiation exists for other kinds ofbacteria which are pathogenic or which are associated with harmfuleffects in humans, such as Salmonella, staphylococci, or Streptococcus.Efficient methodologies to identify and enumerate staphylococci,commonly classified as a Gram-positive coccus bacteria, in food samplesare particularly needed because staphylococcus enterotoxin associatedwith staphylococci contamination of chicken, other processed meats anddairy products is the agent responsible for most common cases of foodpoisoning.

Processes and products which allow for the identification and/orenumeration of such bacteria would provide significant benefit to avariety of industries including, but not limited to, manufacturers offood products, cosmetics and diagnostic assays.

SUMMARY OF THE INVENTION

This invention provides a method of identifying and/or enumeratingstaphylococci in a sample possibly contaminated with bacteria orpotentially containing such bacteria. This method includes the steps ofi) inoculating a selective medium with an aliquot of a sample, whereinthe medium comprises inhibitors to promote the growth of staphylococci,a first substrate capable of producing an observable first color in thepresence of beta-glucosidase and second substrate capable of producing asecond color in the presence of phosphatase, ii) incubating theinoculated medium to produce bacterial colonies of sufficient size toallow visualization of the colonies in the presence of the first andsecond substrates in the medium, and iii) enumerating the coloniesidentified by the presence of the second color of the second substrateto give the number of staphylococci in the sample.

The present invention exploits the herein first reported, empiricalobservation that even though staphylococci produce the enzyme,beta-glucosidase, this genus of bacteria is not able to produce ametabolite that will enzymatically react with5-bromo-4-chloro-3-indolyl-beta-D-glucopyranoside, a substrate commonlyused to detect the presence of beta-glucosidase produced by otherbacteria. In view of this unexpected observation, one embodiment of thepresent invention includes a selective solid medium containinginhibitors to enhance staphylococci growth as well as a firstglucopyranoside substrate, such as5-bromo4-chloro-3-indolyl-beta-D-glucopyranoside, and a secondphosphatase substrate, such as 6-chloro-3-indolylphosphate or5-bromo-6-chloro-3-indolylphosphate. In this embodiment, staphylococciin a sample will produce metabolites that will react with thephosphatase substrate in the medium to produce colonies having red tored-violet color while other bacteria in the sample will producebeta-glucosidase that will react with the glucopyranoside substrate inthe medium and produce colonies having a blue color.

In a particularly preferred embodiment of this invention, a thin filmculture plate device, such as a PETRIFILM thin film culture plate, isprepared using a selective dry culture medium containing nutrients,selected inhibitors, a first glucopyranoside substrate and a secondphosphatase substrate. When the thin film culture plate device isinoculated with a sample and then incubated for a sufficient period oftime, staphylococci in the sample will produce colonies in the culturemedium of the thin film device having a red color and other bacteria inthe sample will produce colonies having a blue color.

The present invention provides an efficient, visual, colorometric methodof identifying and enumerating staphylococci in a sample in a variety ofsolid media.

BRIEF DESCRIPTION OF THE DRAWING

FIG. 1 is a top perspective view, partially in section, of a thin filmculture device used to grow microorganisms.

DETAILED DESCRIPTION

The present invention exploits the herein first reported, empiricalobservation that even though staphylococci produce the enzyme,beta-glucosidase, this genus of bacteria is not able to produce ametabolite that will enzymatically react with5-bromo-4-chloro-3-indolyl-beta-D-glucopyranoside, a substrate commonlyused to detect the presence of beta-glucosidase produced by otherbacteria. In view of this unexpected observation, one embodiment of thepresent invention includes a selective medium containing inhibitors toenhance staphylococci growth as well as a first glucopyranosidesubstrate, such as 5-bromo-4-chloro-3-indolyl-beta-D-glucopyranoside,and a second phosphatase substrate, such as 6-chloro-3-indolylphosphateor 5-bromo-6-chloro-3-indolylphosphate. In this embodiment,staphylococci in a sample will produce metabolites that will react withthe phosphate substrate in the medium to produce colonies having red tored-violet color while other bacteria in the sample will producebeta-glucosidase that will react with the glucopyranoside substrate inthe medium and produce colonies having a blue color.

A preferred selective media includes suitable nutrients, salts and ionsneeded for staphylococci to produce detectable colonies as well asinhibitors to prevent growth of other undesired microorganisms. Suitablenutrients, salts and ions include casein peptone, yeast extract, beefextract, glucose, sodium pyruvate, disodium phosphate, monopotassiumphosphate, ferric ammonium titrate and sodium carbonate. Before use, thelisted components are mixed or blended and then sterilized. Exposure toethylene oxide, for example, is sufficient to sterilize the components.Selected inhibitors are also included in the medium to prevent thegrowth of undesired microorganisms including but not limited toGram-negative bacteria such a E. coli as well as most Gram-positivebacteria. Inhibition of these undesired bacteria allows the growth ofstaphylococci to be selectively enhanced. Suitable inhibitors include avariety of well known antimicrobial or antibiotic compounds. Forexample, quinoline-based antibacterial compounds such as nalidixic acid,polymyxin-derived compounds such as colistin methanesulfonate and saltssuch as lithium chloride are known, preferred inhibitors.

The selective medium of this invention also includes two differentsubstrates which allow visual detection of different types of growingcolonies. One substrate is a glucopyranoside substrate which will reactwith a beta-glucosidase metabolite produced by bacteria, except bacteriaof the genus staphylococci, to produce a detectable substrate.Preferably the detectable substrate is a colored, visually detectablesubstrate. A variety of known beta-glucosidase-indicating compounds arecommercial available. Typical amounts of these substrates which may usedin the selective medium range from about 25-500 mg/ml and preferablyrange from about 25-50 mg/ml. Suitable beta-glucosidase indicatorsinclude 5-bromo-4-chloro-3-indolyl-beta-D-glucopyranoside which providesa blue precipitate in the presence of beta-glucosidase and4-methyhmbelliferyl-β-D-glucopyranoside which provides a detectablefluorescent substrate in the presence of beta-glucosidase.

A second substrate is an indolylphosphatase substrate that will reactwith a phosphatase metabolite produced by staphylococci to produce adetectable, preferably precipitated, substrate. Commercially availablephosphatase indicators include 6-chloro-3-indolylphosphate or5-bromo-6-chloro-3-indolylphosphate which react with staphylocciphosphatase to provide a red to red-violet colored, precipitatedsubstrate. Typically amounts of phosphatase substrates which may be usedin the selective medium range from about 100-500 mg/ml and preferablyrange from about 200-300 mg/ml.

The selective medium of the present invention is generally mixed withgel-forming materials to give a solid medium. A solid medium provides adefined area for growth of bacterial colonies which may be present in asample. Suitable gel-forming materials include commercially availableagar as well as methyl pectin which is described in U.S. Pat. No.5,210,022. Other gelling materials which are preferred for use in thinfilm culture plate devices include gel-forming gums such as xanthan gum,locust bean gum, rhamsan gum and guar gum or mixtures thereof.

When the present selective media is used, a sample is typically dilutedwith a diluent and then used to inoculate the solid medium. Theinoculated medium is then incubated for about 40-56 hours and the mediumis then visually inspected. Staphylococci which are present in a samplewill produce colonies which react with the phosphatase substrate in themedium to provide red to red-violet colored zones while other bacteriain the sample will produce colonies which produce beta-glucosidase thatwill react with the glucopyranoside substrate in the medium and provideblue colored zones.

In a particularly preferred embodiment of this invention, a thin filmculture plate device, essentially similar to an Aerobic Count PETRIFILMculture plate device (commercially available form 3M, St. Paul, Minn.,catalog number 6400) is prepared using a dry culture medium containingselected inhibitors, a first glucopyranoside substrate and a secondphosphate substrate. When the thin film culture plate device isinoculated with a sample and then incubated for a sufficient period oftime, staphylococci in the sample will produce colonies on the thin filmdevice having a red color and other bacteria in the sample will producecolonies on the device having a blue color.

FIG. 1 illustrates a thin film culture plate device suitable for usewith the present invention. Such thin film culture devices are describedin U.S. Pat. Nos. 4,565,783, 5,089,413 and 5,232,838 which are allincorporated by reference in this application for the purposes ofdescribing the processes of making and using these types of thin filmculture plate devices.

Briefly, a dry culture device 10 includes a body member having aself-supporting, waterproof substrate 12. Substrate 12 is preferably arelatively stiff material made of a waterproof polymer that does notabsorb water such as polyester, polypropylene, or polystyrene. Othersuitable waterproof materials include substrates such as papercontaining a waterproof polyethylene coating.

The upper surface of substrate 12 is coated with a layer of adhesive 14,such as isooctyl acrylate/acrylamide in a weight ratio of 96/4 or anaqueous emulsion suspension of a copolymer of isooctyl acrylate andN-vinylpyrrolidone at a 98:2 weight ratio described in U.S. Pat. No.5,232,838 as well as IGEPAL CA 897 nonionic surfactant and lauroylperoxide, which serves to hold a cold-water soluble dry powder 16 thatis a mixture of a dry gelling agent and microbial growth nutrientsadhered in a uniform layer on the substrate. The adhesive must notinhibit the growth of the microorganisms. In addition, the adhesiveshould be sufficiently transparent when hydrated to allow viewing ofbacterial colonies growing on the surface of the substrate through thecoated substrate. The adhesive 14 should also be coated on the substratein a thickness which allows the substrate to be uniformly coated withpowdered gelling agents and nutrients without completely embedding suchparticles in the adhesive.

A layer of cold-water soluble dry powder 16 is uniformly adhered to theadhesive layer 14. Preferably, the soluble powder contains gellingagents and microbial growth nutrients. Suitable gelling agents includeboth natural and synthetic agents which form solutions with water atroom temperature. These gelling agents include hydroxyethylcellulose,carboxymethyl cellulose, polyacrylamide, or algin. A preferred mixtureof gelling agents or gums includes xanthan gum, locust bean gum, rhamsangum and guar gum. Suitable nutrients for use in the present inventionalso include nutrients which are soluble in water at room temperature.The specific types of nutrients are selected in order to promote thegrowth of the microorganisms which will be grown on the substrate 12. Avariety of nutrients may be used which include components such ascarbohydrates, proteins and minerals. A preferred nutrient powderincludes casein peptone, yeast extract, glucose, ferric ammoniumcitrate, sodium pyruvate, sodium carbonate and guar gum mixed with thewater-based copolymer of isooctyl acrylate and N-vinylpyrrolidoneadhesive. Additional components include salts and minerals such assodium pyruvate, monobasic potassium phosphate, dibasic potassiumphosphate as well as inhibitors such as colistin methanesulfonate,lithium chloride and nalidixic acid.

In the device illustrated in FIG. 1, a cover sheet 20 is attached to oneedge of an upper surface of the substrate 12. Cover sheet 20 ispreferably made of a transparent film or sheet material in order tofacilitate counting of bacterial colonies present on the substrate. Inaddition, cover sheet 20 is preferably impermeable to bacteria and watervapor in order to avoid the risk of contamination and deterioration ofthe components. A preferred material for use as a cover sheet 20 isbiaxially-oriented polypropylene.

A layer of a water-insoluble adhesive containing an indicator dye isapplied on the surface of cover sheet 20 that is adjacent to substrate12. Suitable adhesives 22 which are applied to cover sheet 20 includeacrylate based adhesives such as the adhesives described in U.S. Pat.No. 4,565,783 and U.S. Pat. Re No. 24,906 as well as an adhesivecopolymer of isooctyl acrylate and acrylic acid in a 98:2 weight ratio.Preferred adhesives are generally water-insoluble isooctylacrylate-based adhesives which will not detrimentally interfere with, orhamper the growth of, microorganisms such as bacteria. The cover sheetalso includes a beta-glucosidase substrate such as5-bromo-4-chloro-3-indolyl-β-D-glucopyranoside and a phosphatasesubstrate such as 5-bromo-6-chloro-3-indolylphosphate. If desired,additional gelling agents or nutrients may also be adhered to theadhesive that is applied to the surface of cover sheet 20.

In use, a predetermined amount of inoculum, typically about onemilliliter of inoculum, is added to a device illustrated in FIG. 1 bypulling back cover sheet 20 and adding an aqueous test sample or waterto the middle of substrate 12. Cover sheet 20 is then replaced oversubstrate 12 and the inoculum is evenly spread on the substrate using aweighted circular template which is used to confine the inoculum to aspecific area of substrate 12. As the inoculum contacts and is spread onsubstrate 12, the gelling agents and nutrients adhered to substrate 12hydrate to form a growth-supporting nutrient gel. The inoculated deviceis then incubated for a predetermined time after which the number ofbacterial colonies growing on the substrate may be counted through thetransparent cover sheet 20.

The following examples are provided to further illustrate the practiceof various embodiments of the present invention. The examples areprovided for illustrative purposes only and should not be construed tolimit the scope of the invention which is set out in the appendedclaims.

EXAMPLES Example 1

One side of 0.13 mm thick polyethylene-coated paper (Schoeller PaperInc., of Pulaski, N.Y.) was coated with a noninhibitory adhesivecopolymer of isooctyl acrylate (IOA) and acrylamide at a 98:2 weightratio (IOA:ACM) at a level (measured when dry) of 0.93 rag/cm² anddried.

The adhesive was then dusted uniformly with a mixture of 2 parts byweight guar gum (Meyhall Chemical AG, Kreuzlingen, Switzerland) and 1part of a nutrient mixture containing in parts by weight: casein peptone5 parts, yeast extract 2.5 parts, glucose 1 part, sodium pyruvate 10parts, beef extract 1 part, disodium phosphate 2 parts, andmonopotassium phosphate 0.7 parts. This powder-coated paper wasdisinfected by exposure to ethylene oxide.

A cover sheet was made from a sheet of 0.04 mm thick, transparent,biaxially-oriented, corona-treated polypropylene film, coated with anoninhibitory adhesive copolymer of isooctyl acrylate (IOA) and acrylicacid (AA) in a 98:2 weight ratio (IOA:AA), at a level (measured whendry) of 0.93 mg/cm², and dried. The adhesive was then dusted uniformlywith a mixture of 2 parts by weight xanthan gum (KELTROL gum, availablefrom Kelco Inc., San Diego, Calif.), 2 parts locust bean gum (GENU gum,available from Hercules, Inc., Wilmington, Del.), 1 part rhamsan gum(K1A 112 available from Kelco Inc.) and 1 part M150 guar gum (MEYPROGATgum, Meyhall Chemical AG). The excess powder was shaken loose. Thepowders had previously been disinfected by exposure to ethylene oxide orgamma irradiation.

Both the adhesive-coated and powder-coated bottom portion and coversheet were cut into 7.6 cm×10.2 cm pieces, placed together with thepowdered sides facing each other, and heat-sealed together along oneedge.

In use, the device was placed on a level surface, and the top coversheet folded back, exposing the powder-coated surface of the bottomsection of the device. A 1 ml aqueous test sample containing a solution(prepared as described below) of a mixture of substrate and bacteria wascarefully placed in the center of the bottom section of the device, andthe cover sheet replaced, powder-coated side down. A weighted spreaderwas applied to evenly spread the aqueous test sample over thepowder-coated surfaces of the culture media device. The inoculateddevice was placed in an incubator and incubated in the normal manner at35° C. After incubation, the device was read just as with a standardpour-plate.

Solutions of substrate and bacteria were prepared as follows: 10.1 mg of6-chloro-3-indolyl-β-D-glucopyranoside para-toluidine salt was dissolvedin 100 μl of N,N-dimethylformamide. When 400 μl of ethanol was added, aprecipitate formed, therefore 200 μl of additional N,N-dimethylformamidewas added. To 10.2 mg of 6-chloro-3-indolylphosphate para-toluidine saltand 6-chloro-5-bromo-3-indolylphosphate para-toluidine saltrespectively, both dissolved in 300 μl of N,N-dimethylformamide wasadded 400 and 200 μl of ethanol, respectively. 9.9 mg of5-bromo-4-chloro-3-indolyl-β-D-glucopyranoside para-toluidine salt(available from Biosynth International Inc., Skokie, Ill.) was dissolvedin 200 μl of N,N-dimethylformamide and 300 μl of ethanol.

Three colonies each of Staphylococcus aureus and Streptococcus faecalisgrowing on sheep's blood agar plates and derived from cultures ATCC29213 and ATCC 29212 respectively were picked with the PROMPTinoculation system No. 6314 (available from Baxter Scientific Products,Catalog No. B1026-10D) to provide bacterial suspensions which weresubsequently diluted 1/10,000 with Butterfield's buffer.

Four 10 ml portions of each bacterial suspension in buffer weretransferred into each of 4 sterile tubes to provide a total of 8 tubes.To separate tubes of each bacterium was added an aliquot of thesubstrate solution prepared above as shown below:

140 μl of 6-chloro-3-indolyl-β-D-glucopyranoside salt (substrate A)

100 μl of 5-bromo-4-chloro-3-indolyl-β-D-glucopyranoside salt (substrateB)

140 μl of 6-chloro-3-indolylphosphate salt (substrate C) and

100 μl of 6-chloro-5-bromo-3-indolylphosphate salt (substrate D)

Four mixtures were prepared by combining 1 ml of the S. aureus and6-chloro-3-indolylphosphate mixture with 1 ml of the S. faecalis and5-bromo-4-chloro-3-indolyl-β-D-glucopyranoside mixture; 1 ml of the S.aureus and 6-chloro-5-bromo-3-indolylphosphate mixture with 1 ml of theS. faecalis and 5-bromo-4-chloro-3-indolyl-β-D-glucopyranoside mixture;1 ml of the S. faecalis and 6-chloro-3-indolylphosphate mixture with 1ml of the S. aureus and 5-bromo-4-chloro-3-indolyl-β-D-glucopyranosidemixture and 1 ml of the S. faecalis and6-chloro-5-bromo-3-indolylphosphate mixture with 1 ml of the S. aureusand 5-bromo-4-chloro-3-indolyl-β-D-glucopyranoside mixture. It wasobserved that all of the mixtures showed some cloudiness, indicatingprecipitation.

One milliliter of each of the twelve substrate-bacteria suspensions wasused to inoculate twelve of the culture media devices prepared asdescribed above. The devices were incubated at 35° C. The results wereobserved after 24 hours of incubation.

                  TABLE 1                                                         ______________________________________                                        Bacteria    Substrate                                                                              Results                                                  ______________________________________                                        S. aureus   A        No colonies observed                                     S. aureus   D        Dark compact purple colonies                             S. aureus   C        Lighter more diffuse purple                                                   colonies                                                 S. aureus   B        No colonies observed                                     S. faecalis C        Pale diffuse purple colonies                             S. faecalis D        Slightly darker more compact                                                  purple colonies                                          S. faecalis A        No colonies observed                                     S. faecalis B        Intense somewhat diffuse blue-                                                green colonies                                           S. aureus/S. faecalis                                                                     C, B     Mixed blue-green & purple                                                     colonies                                                 S. aureus/S. faecalis                                                                     D, B     Mixed blue-green & purple                                                     colonies                                                 S. aureus/S. faecalis                                                                     B, C     Mixed blue-green & purple                                                     colonies                                                 S. aureus/S. faecalis                                                                     B, D     Mixed blue-green & purple                                                     colonies                                                 ______________________________________                                    

Surprisingly, the S. aureus did not metabolize the indicator when thesubstrate was solely a glucopyranoside as shown by the lack of coloredcolonies. The S. faecalis produced colored colonies on the5-bromo-4-chloro-3-indolyl-β-D-glucopyranoside substrate, indicating aclear difference in the ability of these bacteria to use that substrate.

Example 2

Demonstration of Lack of beta-Glucosidase Activity by StaphylococcusVariants

Staphyococcus aureus variants were obtained from the Centers For DiseaseControl, Atlanta, Ga. and maintained as part of a private collection atMinnesota Mining and Manufacturing Company (3M, St. Paul, Minn.) weretested for β-D-glucosidase activity using three systems, agar, broth andPETRIFILM thin film culture medium devices (described in Example 1). Theagar contained 5-bromo-4-chloro-3-indolyl-β-D-glucopyranoside(x-glucoside) as the substrate. The conventional buffered peptone broth(available from Difco, Detroit, Mich.) was carbohydrate free and wastested using 5-bromo-4-chloro-3-indolyl-β-D-glucopyranoside or4-methylumbelliferyl-β-D-glucopyranoside (4-MUG) (solutions prepared asdescribed in Example 1) as substrates. The PETRIFILM culture platedevices were prepared as described in Example 1 and were tested byplating as described in Example 1 on the culture plate devices usingeither x-glucoside and 6-chloro-5-bromo-3-indolylphosphatepara-toluidine salt (phosphate)(solutions prepared as described inExample 1) as substrates. As shown in Table 2, S. aureus variants grownon the agar plates failed to use the x-glucoside substrate, the coloniesremaining off-white to golden rather than turning the intense blue seenwith Streptococci although the same variants all used the 4-MUGsubstrate. All 32 S. aureus variants in broth metabolized the 4-MUGsubstrate but failed to metabolize the x-glucoside substrate. All 32 S.aureus variants grew as purple colonies on the PETRIFILM culture platedevices using 6-chloro-5-bromo-3-indolylphosphate substrate but failingto metabolize the x-glucoside substrate.

The identity of both purple and blue-green colonies present on thePETRIFILM culture plate devices was confirmed by streaking colonies ofeach bacterium to sheep's blood agar plates and reidentifying the purecultures of bacteria by conventional methods.

                                      TABLE 2                                     __________________________________________________________________________                Broth with                                                                          Broth with                                                                           Agar with                                                                           Agar with                                                                            Petrifilm with                                                                       Petrifilm with                               4-MUG X-Glucoside                                                                          4-MUG X-Glucoside                                                                          4-MUG  X-glucoside                      Organism    Substrate                                                                           Substrate                                                                            Substrate                                                                           Substrate                                                                            Substrate                                                                            Substrate                        __________________________________________________________________________    S. faecalis control (88)                                                                  +     +      NA    NA     +      +                                coagulase negative                                                                        -     -      -/+*         -      -                                Staph control                                                                 S. aureus - +     -      + (weak)                                                                            -      +      -                                76, 77, 78, 79, 80, 81,                                                       82, 83, 84, 85, 86, 87,                                                       1043, 1052, 1054,                                                             1060, 1068, 1070,                                                             1072, 1078, 1081,                                                             1112, 1117, 1119,                                                             1120, 1154, 1155,                                                             1156, 1166, 1167,                                                             1168, 2013                                                                    Entero-coccus controls                                                                    NA    NA     all + all +  NA     NA                               (10 strains)                                                                  __________________________________________________________________________     *10 strains were used, 8 were negative, 2 were positive                  

Example 3

Using thin film culture media devices prepared as described in Example1, the presence of staphylococci in foods was evaluated in comparisonwith Baird-Parker agar as described below.

Stressed staphylococci were prepared to simulate actual "real life"conditions by growing Staphylococcus aureus strain 1060 overnight intrypticase soy broth (available from BBL Microbiological Systems,Cockeyesville, Md.). A 0.01 ml sample of this broth culture was pipettedinto 10 ml of 0.1M, pH 7.2 phosphate buffer prewarmed to 52° C. Thismixture was incubated at 52° C. for 20 minutes, then diluted withButterfield's buffer for plating. The dilution used was 1:100(mixture:buffer). This buffer was stored at 4° C. then warmed to roomtemperature and used hereinafter.

The water-based adhesive composition to be coated on polyethylene paperas described in Example 1 was formed by dissolving 500 g of nutrientpowder consisting of 132 parts of casein peptone, 66 parts of yeastextract, 26 parts of glucose, 5.8 parts of ferric ammonium citrate, 264parts of sodium pyruvate and 5.8 parts of sodium carbonate plus 75 g ofM150 guar gum in 5 kg of adhesive. The adhesive similar to thatdescribed in Example 1 was an aqueous emulsion suspension of a copolymerof isooctyl acrylate (IOA) and N-vinylpyrrolidone (NVP) at a 98:2 weightratio (IOA:NVP) with IGEPAL CA 897 nonionic surfactant and lauroylperoxide to which had been added a stirred aqueous solution of 0.2503parts colistin methanesulfonate, 175 parts of lithium chloride and 2450parts of distilled water to which had been added a solution of 0.4001parts of nalidixic acid which had been dissolved in 50 parts water bythe addition of a small amount of 10N sodium hydroxide solution and0.075 parts of methyl-β-D-glucoside.. The coated substrate was dried inan air oven at about 90° C. to yield a sticky layer on the surface ofthe substrate at a dry weight of 3.55 to 4.2 mg/cm².

A mixture of cold-water-soluble powders including one part nutrientpowder and two parts mixed gums powder formed of proportions by weightof 2 parts of xanthan gum (KELTROL gum, 2 parts of locus bean gum(MYPRODYNE gum), 1 part of K1A 112 and 1 part of M150 guar gum, wasdusted over the surface of the water-based adhesive layer. Any excesspowder was shaken loose. This adhesive-coated and powder-coated paperwas used to form the bottom portion of the culture media device.

Cover sheets were made from sheets of 0.04 mm thick, transparent,biaxially-oriented, corona-treated polypropylene film, coated with anadhesive copolymer of isooctyl acrylate (IOA) and acrylic acid (AA) in a98:2 weight ratio (IOA:AA), at a level (measured when dry) of 0.969 to1.29 mg/cm², and containing 0.66 g of5-bromo-4-chloro-3-indolyl-β-D-glucopyranoside and 5.43 g of5-bromo-6-chloro-3-indolylphosphate in 1.2 liters of methanol and 1.2liters of acetone per 3 kg of adhesive polymer and the adhesivecomposition was dried. The cover sheets were dusted with the mixed gumspowder.

A mixture of 9 ml of S. aureus in Butterfield's buffer and 1 ml of foodsample was prepared to be used to inoculate the culture plate devices.The foods used were chicken and cheddar, Monterey jack, swiss andmozzarella cheeses diluted 1 to 10 with water. Duplicate 1 ml portionswere applied to culture plate devices, quadruplicate 0.5 ml portionswere applied to freshly prepared Baird-Parker agar. Undiluted pure S.aureus and S. aureus diluted to 1:10 were used to inoculate positivecontrol culture plates. All of the samples were incubated at 35° C. andmedia counts were taken at 40 hours. The results are summarized in Table3.

                  TABLE 3                                                         ______________________________________                                        Mean Counts (cfu/ml)                                                                                        Productivity                                             Baird-Parker                                                                           Magenta PO.sub.4 *                                                                        Magenta                                         ______________________________________                                        Pure Staph-10.sup.o                                                                      144.5      163.5       113%                                        Pure Staph-10.sup.-1                                                                     15.5       13           84%                                        Cheddar    173.5      212         122%                                        Swiss      160        173.5       108%                                        Mozzarella 193        195         101%                                        Monterey Jack                                                                            196.5      191          97%                                        Chicken    226        195.5        87%                                        ______________________________________                                         *5-bromo-6-chloro-3-indolylphosphate gives a bright magenta color and is      preferred phosphate substrate.                                           

Productivity is a comparison of the method and device of the inventionto the Baird-Parker agar, a well-known standard. S. aureus colonies arebright magenta in the device of the invention. The results show that thenumber of colony forming units are about the same using both methods.

I claim:
 1. A thin film culture plate device for identifying andenumerating staphylococci in a sample containing more than one speciesof bacteria, comprising:i) a self-supporting, waterproof substrate towhich is adhered a rehydratable selective medium powder containingnutrients, inhibitors to promote the growth of staphylococci, and atleast one gelling agent, and ii) a transparent cover sheet having alayer of adhesive consisting essentially of a dual substrate systemhaving an indolylglucopyranoside substrate which provides a visiblefirst color change in the presence of beta-glucosidase and a phosphatesubstrate which provides a visible second color change in the presenceof staphylococci and at least one gelling agent.
 2. The device of claim1 wherein the inhibitors are selected from the group consisting ofcolistin methanesulfonate, nalidixic acid and lithium chloride.
 3. Thedevice of claim 1 wherein the indolylglucopyranoside substrate is5-bromo-4-chloro-3-indolyl-beta-D-glucopyranoside.
 4. The device ofclaim 1 wherein the phosphate is selected from the group consisting of6-chloro-3-indolylphosphate and 5-bromo-6-chloro-3-indolylphosphate. 5.The device of claim 1 wherein the gelling agent is selected from groupconsisting of xanthan gum, locust bean gum, rhamsan gum and guar gum.